EP3706797A1 - Composition for the treatment of intracellular bacterial infection - Google Patents

Composition for the treatment of intracellular bacterial infection

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Publication number
EP3706797A1
EP3706797A1 EP18800925.2A EP18800925A EP3706797A1 EP 3706797 A1 EP3706797 A1 EP 3706797A1 EP 18800925 A EP18800925 A EP 18800925A EP 3706797 A1 EP3706797 A1 EP 3706797A1
Authority
EP
European Patent Office
Prior art keywords
cell
agent
antibacterial agent
cells
photosensitizing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18800925.2A
Other languages
German (de)
English (en)
French (fr)
Inventor
Anders HØGSET
Sebastian A.J. ZAAT
Xiaolin Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Stichting Amsterdam Umc
PCI Biotech AS
Original Assignee
Academisch Medisch Centrum
PCI Biotech AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Academisch Medisch Centrum, PCI Biotech AS filed Critical Academisch Medisch Centrum
Publication of EP3706797A1 publication Critical patent/EP3706797A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method of treating intracellular bacterial infections using a photochemical internalization method to introduce an antibacterial agent to kill the bacteria.
  • the methods have particular utility in treating hard to treat infections such as those which occur in biomaterial-associated infections.
  • tuberculosis Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus
  • Staphylococcus aureus is a notorious cause of many infectious diseases (Abed & Couvreur, 2014, Int. J. Antimicrob. Ag., 43(6), p485-496; Xiong et al., 2014, Adv. Drug Deliver. Rev., 7, p63-76; Briones et al., 2008, J. Control Release, 125(3), p210-227).
  • infectious diseases Abed & Couvreur, 2014, Int. J. Antimicrob. Ag., 43(6), p485-496; Xiong et al., 2014, Adv. Drug Deliver. Rev., 7, p63-76; Briones et al., 2008, J. Control Release, 125(3), p210-227).
  • aureus may cause a wide variety of skin or tissue infections as well as life-threatening invasive diseases such as bacteremia, biomaterial-associated infection, diabetic foot infection, endocarditis and osteomyelitis (Lew & Waldvogel, 2004, Lancet, 364(9431 ), p369-379; Busscher et ai, 2012, Sci. Transl. Med., 4(153); von Eiff et al, 2002, Lancet Infect. Dis., 2(1 1 ), p677-685; Lipsky et al., 2004, Clin. Infect. Dis., 39(7), p885-910; Saginur & Suh, 2008, Int. J. Antimicrob. Agents., 32 Suppl 1 , S21 -25; Dhawan et al., 1998, Infect. Immun., 66(7), p3476- 3479).
  • bacteremia biomaterial-associated infection
  • diabetic foot infection diabetic foot infection
  • aureus and S. epidermidis or other bacterial species may cause the occurrence or recurrence of intracellular infection-associated diseases. It is furthermore known that some bacteria remain in phagosomes and prevent fusion of these phagosomes with lysosomes. Examples of bacteria which adopt this strategy include Mycobacterium tuberculosis (the cause of the devastating and widespread disease of tuberculosis) and Coxiella burnetii (the cause of Q-fever).
  • Intracellular infection is usually very difficult to treat, since the majority of existing antibiotics has limited intracellular activity (Abed & Couvreur, 2014 supra; Xiong et al., 2014, supra; Briones et al., 2008, supra; Tulkens, 1991 , Eur. J. Clin. Microbiol., 10(2), p100-106 and Carryn et al., 2003, Infect. Dis. Clin. N. Am., 17(3), p615-634).
  • Beta-lactam antibiotics and aminoglycosides have low penetration into eukaryotic cells (Abed & Couvreur, 2014, supra; Tulkens, 1991 , supra; Carryn et al., 2003, supra; Serai et al., 2003, J. Antimicrob. Chemoth., 51 (5), p1 167-1 173; and Carryn et al., 2002, Antimicrob. Agents Ch., 46(7), p2095-2103), and although fluoroquinolones and macrolides can pass the eukaryotic cell membrane, they show low intracellular retention (Serai et al., 2003, supra; and Serai et al., 2003, Antimicrob.
  • Rifampicin has good intracellular penetrating capacity, but is subject to very high frequencies of resistance development when used as single antibiotic. Therefore combination therapy of rifampicin with other antibiotics is mandatory (Forrest & Tamura, 2010, Clin.
  • PDT antibacterial photodynamic therapy
  • PDT therapy is based on the employment of photosensitive agents
  • PS photosensitizers, PS
  • PS can accumulate in the cytoplasmic membrane of bacteria and actively kill them by the induced reactive oxygen reaction upon illumination of the site of treatment. However, only bacteria in the direct vicinity of the photosensitizer are killed.
  • the present invention provides a new method for killing intracellular bacteria which involves the use of photochemical internalization (PCI) and an antibacterial agent.
  • PCI photochemical internalization
  • photosensitizers specifically localize in membranes of endocytic vesicles and disrupt these membranes (partially) upon illumination, causing cytosolic release of molecules.
  • PCI of antibacterial agents provides a simple mechanism for killing intracellular bacteria.
  • PCI methods provide a mechanism for introducing molecules into the cytosol of a cell in a manner which does not result in widespread cell destruction or cell death if the methodology is suitably adjusted to avoid excessive toxic species production, e.g. by lowering illumination times or photosensitizer dose.
  • the basic method of photochemical internalisation (PCI) is described in WO 96/07432 and WO 00/54802, which are incorporated herein by reference. In such methods, the molecule to be internalised (which in the present invention would be the
  • a photosensitizing agent e.g. a lysosome or endosome.
  • an intracellular vesicle e.g. a lysosome or endosome.
  • the photosensitizing agent is activated which directly or indirectly generates reactive oxygen species which disrupt the intracellular vesicle's membranes. This allows the internalized molecule to be released into the cytosol. It was found that in such a method the functionality or the viability of the majority of the cells was not deleteriously affected.
  • This method of the invention is particularly advantageous because it is not a complex method and may be used with a variety of antibacterial agents and different target bacteria. It also allows the use of lower concentrations of the antibacterial agent than is required for conventional methods, whilst achieving effective antibiotic effects. This prevents resistance development. Furthermore, the timing and location of irradiation to release the molecules may be controlled such that it is released only at the time and location that is desired to achieve the required effects. As such, exposure of cells to the various components is minimised, and undesirable side effects are minimised. This is in contrast to the standard techniques for antibacterial treatment, where it is not possible to control the timing and location of the release of the various components and high concentrations of the various components and/or their carriers are needed.
  • Infectious diseases associated with intracellular survival of bacterial pathogens can occur or relapse at different sites of the human body (e.g. skin, deep tissues, urinary tract and lung).
  • nonprofessional phagocytic cells such as epithelial cells, endothelial cells, osteoblasts and fibroblasts also can be niches of intracellular bacteria.
  • the PCI method of the invention may be achieved at a specific location by applying light at the site of infection or at the location of the cells in which intracellular bacteria reside.
  • the method may be used for treatment of (sub)cutaneous skin or mucosal infections/damages such as chronic wounds, ulcers, abscesses and diabetic foot infection as well as oral and nasal infections such as chronic rhinosinusitis and periodontitis, where the site of infection is relatively accessible for light.
  • Infection in internal organs (e.g. lungs) or in deep tissue areas may be treated using light administered by fiber optic devices, for example.
  • PCI photochemical internalization
  • PS photosensitizers
  • the PS localizes in vesicle membranes during the formation of endosomes/phagosomes containing the antibacterial agent.
  • the antibacterial agents are released by activation of the PS (by disrupting the membranes of endosomes/phagosomes upon illumination) into the cytosol where they may come into contact with bacteria (present by co- internalization or pre-existing in the cells).
  • the PS are also dissociated from the membrane and may be re-located to other endosomes/phagosomes that may contain antibacterial agents and/or bacteria during the illumination period thereby causing release of the bacteria/antibacterial agents in those
  • the present invention provides a method of treating or preventing an intracellular bacterial infection, comprising contacting the cell(s) which are infected with an antibacterial agent and a photosensitizing agent and irradiating the cell(s) with light of a wavelength effective to activate the
  • the antibacterial agent is released into the cytosol of the cell(s) and kills, damages or prevents the replication of bacteria in said cell(s).
  • This method may be performed in vitro, but is preferably performed in vivo and said cell(s) is in a subject.
  • the method prevent the development of bacterial resistance. They may be used against antibacterial or antibiotic resistant bacteria.
  • the photosensitizing agent and the antibacterial agent are each taken up into an intracellular vesicle; and when the cell is irradiated the membrane of the intracellular vesicle is disrupted releasing the molecules into the cytosol of the cell.
  • the different components may be taken up into the same or a different intracellular vesicle relative to each other. It has been found that active species produced by photosensitizers may extend beyond the vesicle in which they are contained and/or that vesicles may coalesce allowing the contents of a vesicle to be released by coalescing with a disrupted vesicle.
  • take up signifies that the molecule taken up is wholly contained within the vesicle.
  • the intracellular vesicle is bounded by membranes and may be any such vesicle resulting after endocytosis/phagocytosis, e.g. an endosome, lysosome or phagosome.
  • a "disrupted" compartment refers to destruction of the integrity of the membrane of that compartment either permanently or temporarily, sufficient to allow release of the molecules contained within it.
  • treatment refers to reducing, alleviating or eliminating one or more symptoms of the bacterial infection which is being treated, relative to the symptoms prior to treatment. Such symptoms may be correlated with the abundance of bacteria present in the treated cells and/or on the treated patient or subject.
  • Treatment in an in vitro method comprises killing, damaging or preventing replication of the bacteria in the cell(s) and may be determined by assessing the abundance of viable bacteria in the cell(s).
  • prevention or preventing or prophylaxis refers to delaying or preventing the onset of the symptoms of the bacterial infection. Prevention may be absolute (such that no bacterial infection occurs) or may be effective only in some individuals, or cells, or for a limited amount of time.
  • bacterial infection is invasion of a cell(s) or bodily tissue by bacteria that proliferate at that site and which may result in injury to that cell or tissue.
  • a cell which is "infected” contains one or more intracellular bacteria capable of survival and potentially replication in that cell.
  • the bacterial infection is caused by the bacteria described hereinafter, preferably by bacteria selected from the genera Mycobacterium, Pseudomonas, Escherichia and
  • Staphylococcus also preferred are intracellular bacterial infections by bacteria selected from the genera Coxiella, Listeria, Francisella and Rickettsia. In a preferred aspect the bacteria does not produce spores. In a further preferred aspect the bacteria which appear intracellulararly are not also present in a biofilm, though in an alternative option they may so appear.
  • the infection is present in cells of, or associated with, bones, blood, the heart, urinary tract, lung, skin or mucosal surfaces.
  • bacterial infections to be treated include osteomyelitis, bacteremia, tuberculosis, Q-fever and endocarditis and
  • the method or use is for treating a biomaterial- associated infection and the cells to be treated are present on, or adjacent to, biomaterial introduced into the subject.
  • the biomaterial-associated infection may be peri-implantitis (infection around dental implants).
  • An intracellular bacterial infection which remains after a biomaterial has been removed from a subject may also be treated.
  • Infection is a frequent complication of the use of biomaterials such as medical devices despite all due care being taken in their insertion and care. Infections with Staphylococcus aureus and Staphylococcus epidermidis are particularly prevalent. Infections of biomaterials are very resistant to treatment.
  • Macrophages have been implicated as the host of the intracellular bacteria and maintain a reservoir of bacteria for repeat infections.
  • the methods and uses of the invention allow this "hiding" bacteria to be targeted.
  • biomaterial is an artificial material or device which may be introduced into a subject for curative purposes.
  • biomaterials include a medical device, instrument, implement or equipment, a prosthetic or material, tissue or wound dressing (e.g. for orthopaedic, cardiovascular, urinary tract, surgical, gynaecological or dental purposes).
  • Medical devices include pacemakers, heart valves and stents, medical implements include catheters, prosthetics or material include artificial joints, implants (including breast and dental implants), bone fixation plates and screws and scaffold material (e.g. surgical meshes).
  • Wound dressings include plasters and bandages as well as cements, glues or matrices which may be used for wound repair.
  • the antibacterial agent and/or the photosensitizing agent may be provided on or within (e.g. embedded within or impregnated in) the biomaterial.
  • intracellular bacterial infection refers to an infection in which the bacteria are taken up into the cell and are able to survive and replicate in that cell.
  • the bacteria may additionally exist and replicate outside the cell.
  • intracellular bacteria may be present at any location within the cell, e.g. within the cytosol or in a membrane-contained subcompartment such as a lysosome, endosome or phagosome.
  • Examples of intracellular bacterial infections which may be treated or prevented according to the invention include infections by Mycobacterium (e.g. M. tuberculosis), Pseudomonas (e.g. P. aeruginosa), Escherichia (e.g. E. coli), and Staphylococcus (e.g. S.
  • the subject to be treated is a cow and the bacterial infection is S. aureus mastitis.
  • Other intracellular bacterial infections of interest include infections by Listeria (e.g. Listeria monocytogenes), Francisella (e.g. Francisella tularensis), Coxiella (e.g. C. burnetii) and Rickettsia.
  • the "cell” or “cells” may be in a culture or in a tissue, organ or body.
  • the cell may be provided in vitro, ex vivo or may be within a subject or organism, e.g. an in vivo cell.
  • the term “cell” includes all eukaryotic cells (including insect cells and fungal cells).
  • Representative “cells” thus include all types of mammalian and non-mammalian animal cells, plant cells, insect cells, fungal cells and protozoa.
  • the cells are eukaryotic, e.g. from a mammal, reptile, bird, insect or fish.
  • the cell is from a mammal, particularly a primate, domestic animal, livestock or laboratory animal.
  • the cells are mammalian, for example cells from monkeys, cats, dogs, horses, donkeys, sheep, pigs, goats, cows, mice, rats, rabbits, guinea pigs, but most preferably from humans.
  • the cells which are infected may be phagocytic cells such as macrophages, dendritic cells or neutrophils or may be "non-professional" phagocytic cells such as epithelial cells, endothelial cells, keratinocytes, osteoblasts and fibroblasts.
  • an "antibacterial agent” is an entity (e.g. a molecule) which has the ability to kill, damage or prevent the replication of selected bacteria under in vitro conditions, e.g. when in direct contact under culture conditions.
  • the bacteria are preferably as described herein.
  • bacteria are referred to in both the singular and plural. In particular they are referred to in the singular when defining the type of bacteria to be targeted (i.e. the type, e.g. species, applicable to each bacterium) and in the plural when referring to the treatment to which they may be subjected (i.e. treatment of multiple microorganisms).
  • the antibacterial activity is assessed by determining the MIC or MBC value against one or more bacteria, e.g.
  • Antibacterial activity may be determined by reference to the MIC value, minimum inhibition concentration (MIC), which is defined as the minimum concentration of an antimicrobial agent that inhibits visible growth of micro-organisms in specified liquid media after overnight incubation.
  • MIC minimum inhibition concentration
  • MMC minimum bactericidal concentration
  • antibacterial agents have a MIC value of less than 50 ⁇ g/ml, preferably less than 30 ⁇ g/ml, especially preferably less than 10, 5 or 1 ⁇ g/ml, preferably against a bacteria as described herein.
  • the antibacterial agent is an antibiotic, i.e. selectively treats specific bacteria (genera or species).
  • the antibacterial agent may be selected from aminoglycosides (e.g. amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin or spectinomycin); ansamycins (e.g. geldanamycin, herbimycin or rifaximin); carbacephems (e.g. loracarbef); carbapenems (e.g. ertapenem, doripenem, imipenem, meropenem); cephalosporins (e.g. cefadroxil, cefazolin, cefalexin, cefaclor, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren,
  • aminoglycosides e.g. amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin,
  • cefoperazone cefotaxime, cefpodoxime, ceftazidime, ceftiaxone, cefepime, ceftaroline fosamil or ceftobiprole
  • glycopeptides e.g. teicoplanin, vancomycin, telavancin, dalbavancin or oritavancin
  • lincosamides e.g. clindamycin or lincomycin
  • lipopeptides e.g. daptomycin
  • macrolides e.g.
  • spiramycin azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, fidaxomicin or spiramycin); monobactams (e.g. aztreonam); nitrofurans (e.g. furzolidone or nitrofurantoin); oxazolidinones (e.g. linezolid, posizolid, radezolid or torezolid); penicillins (e.g.
  • ciprofloxacin enfloxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin or temafloxacin); sulfonamides (e.g. mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole,
  • sulfonamides e.g. mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole,
  • sulfamethoxazole sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim- sulfamethoxazole or sulfonamidochrysoidine
  • tetracyclines e.g.
  • the antibacterial agent is an aminoglycoside (preferably gentamicin), a glycopeptide (preferably vancomycin) or a macrolide (preferably as described hereinbefore).
  • gentamicin a glycopeptide
  • macrolide preferably as described hereinbefore.
  • more than one antibacterial agent may be used, e.g. gentamicin and rifampicin.
  • more than one agent e.g. two or three agents
  • they can be used (e.g. administered) simultaneously, sequentially or separately as described herein for the methods and uses of the invention.
  • a “photosensitizing agent” as referred to herein is a compound that is capable of translating the energy of absorbed light into chemical reactions when the agent is activated on illumination at an appropriate wavelength and intensity to generate an activated species.
  • the highly reactive end products of these processes can result in cyto- and vascular toxicity.
  • a photosensitizing agent may be one which localises to intracellular compartments, particularly endosomes, phagosomes or lysosomes.
  • Photosensitizing agents may exert their effects by a variety of mechanisms, directly or indirectly. Thus for example, certain photosensitizing agents become directly toxic when activated by light, whereas others act to generate toxic species, e.g. oxidising agents such as singlet oxygen or other reactive oxygen species, which are extremely destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids.
  • oxidising agents such as singlet oxygen or other reactive oxygen species
  • photosensitizing agents are known in the art and are described in the literature, including in WO96/07432, which is incorporated herein by reference, and may be used in methods and uses of the invention.
  • photosensitizing agents including porphyrins, phthalocyanines and chlorins, (Berg et al., 1997, J. Photochemistry and Photobiology, 65, p403-409, incorporated herein by reference).
  • Other photosensitizing agents include bacteriochlorins.
  • Porphyrins are the most extensively studied photosensitizing agents. Their molecular structure includes four pyrrole rings linked together via methine bridges. They are natural compounds which are often capable of forming metal-complexes. For example in the case of the oxygen transport protein hemoglobin, an iron atom is introduced into the porphyrin core of heme B.
  • Chlorins are large heterocyclic aromatic rings consisting, at the core, of three pyrroles and one pyrroline coupled through four methine linkages. Unlike porphyrin, a chlorin is therefore largely aromatic, but not aromatic through the entire circumference of the ring.
  • the photosensitizing agent is preferably an agent which is taken up into the internal compartments of lysosomes, phagosomes or endosomes.
  • the photosensitizing agent is taken up into intracellular compartments by endocytosis or phagocytosis.
  • photosensitizing agents are amphiphilic photosensitizers (e.g. disulphonated photosensitizing agents) such as amphiphilic phthalocyanines, porphyrins, chlorins, and/or bacteriochlorins, and in particular include sulfonated (preferably
  • the photosensitizing agent is selected from a porphyrin, phthalocyanine, purpurin, chlorin, benzoporphyrin, lysomotropic weak base, naphthalocyanine, cationic dye, tetracycline, 5-aminolevulinic acid and/or esters thereof, or a derivative of any of said agents, preferably TPPS 4 , TPPS 2a , AIPcS 2a , TPCS 2a , 5-aminolevulinic acid or esters of 5-aminolevulinic acids, or pharmaceutically acceptable salts thereof.
  • TPPS 2a tetraphenylporphine disulfonate
  • AIPcS 2a aluminium phthalocyanine disulfonate
  • TPCS 2a tetraphenyl chlorin disulfonate
  • TPBS 2a tetraphenyl bacteriochlorin disulfonate
  • the photosensitizing agent is TPCS 2a (Disulfonated tetraphenyl chlorin, e.g. Amphinex ®).
  • a and/or B includes the options i) A, ii) B or iii) A and B.
  • the arrow indicates the structural difference between the two molecules.
  • the photosensitizing agent may be attached to or associated with or conjugated to one or more carrier molecules or targeting molecules which can act to facilitate or increase the uptake of the photosensitizing agent.
  • the photosensitizing agent may be linked to a carrier.
  • photosensitizing agent may be provided in the form of a conjugate, e.g. a chitosan- based conjugate, for example a conjugate disclosed in WO2013/189663, which is hereby incorporated by reference.
  • a conjugate e.g. a chitosan- based conjugate, for example a conjugate disclosed in WO2013/189663, which is hereby incorporated by reference.
  • contacting refers to bringing the cell(s) and the various components (or agents) used in the method into physical contact with one another under conditions appropriate for internalization into the cells, e.g. preferably at 37°C in an appropriate nutritional medium, e.g. from 25-39°C, or in vivo.
  • the cell may be contacted with the photosensitizing agent and the antibacterial agent used in the method as defined herein sequentially or simultaneously.
  • the components are contacted with the cell(s) simultaneously and preferably are applied to the cell(s) together as described in more detail hereinafter.
  • the different components may be taken up by the cell(s) into the same or different intracellular compartments (e.g. they may be co-translocated). However, in an alternative embodiment the components are not applied together, i.e. are applied at a different time and/or by a different administration route.
  • the cells are then exposed to light of suitable wavelengths to activate the photosensitizing compound which in turn leads to the disruption of the intracellular compartment membranes.
  • WO 02/44396 (which is incorporated herein by reference) describes a method in which the order of the steps in the method may be arranged such that for example the photosensitizing agent is contacted with the cells and activated by irradiation before the molecule to be internalised (in this case the antibacterial agent) is brought into contact with the cells.
  • This method takes advantage of the fact that it is not necessary for the molecule to be internalised to be present in the same cellular subcompartment as the photosensitizing agent at the time of irradiation.
  • said photosensitizing agent and said antibacterial agent as defined herein are applied to the cell together, or separately relative to one another. Irradiation is then performed at a time when the photosensitizing agent and the antibacterial agent appear in the same intracellular compartment. This is referred to as a "light after" method.
  • said method can be performed by contacting said cell with the photosensitizing agent first, followed by contact with the antibacterial agent to be used as defined herein, and irradiation is performed after uptake of the photosensitizing agent into an intracellular compartment, but prior to the cellular uptake of the antibacterial agent into an intracellular compartment containing said photosensitizing agent (e.g. they may be present in a different intracellular compartment at the time of light exposure), preferably prior to cellular uptake into any intracellular compartment, e.g. prior to any cellular uptake.
  • the photosensitizing agent may be administered followed by irradiation and then administration of the antibacterial agent. This is the so-called "light before" method.
  • Internalisation refers to the intracellular, e.g. cytosolic, delivery of molecules.
  • internalisation may include the step of release of molecules from intracellular/membrane bound compartments into the cytosol of the cells.
  • cellular uptake or “translocation” refers to one of the steps of internalisation in which molecules external to the cell membrane are taken into the cell such that they are found interior to the outer lying cell membrane, e.g. by endocytosis, phagocytosis or other appropriate uptake mechanisms, for example into or associated with intracellular membrane-restricted compartments, for example the endoplasmic reticulum, Golgi body, lysosomes, endosomes, phagosomes etc.
  • the method is performed on a cell(s) that is already infected with bacteria.
  • the method may also be performed on a cell(s) that is not yet infected with bacteria or a cell(s) which is in the process of being infected with bacteria (in which case the method is a prevention method).
  • bacteria may be taken up with the photosensitizing agent and/or antibacterial agent during the PCI method.
  • the cell(s) may be a cell(s) which is identified to be at risk of infection (e.g. at a site where intracellular infection is likely, e.g. biomaterial- associated infection when a device is to be used). In this case the method may be performed at the site of interest immediately before placement of the device.
  • the step of contacting the cells with the different agents may be carried out in any convenient or desired way.
  • the cells may conveniently be maintained in an aqueous medium, such as for example appropriate cell culture medium, and at the appropriate time point the various agents can simply be added to the medium under appropriate conditions, for example at an appropriate concentration and for an appropriate length of time.
  • the cells may be contacted with the agents in the presence of serum- free medium, or with serum-containing medium.
  • the application of the agents used in the methods and uses of the invention may be to cells in vitro or in vivo. In the latter case, the application may be via direct (i.e. localized or topical) or indirect (i.e. systemic or non-localized) administration as described in more detail hereinbelow.
  • the photosensitizing agent is brought into contact with the cells at an appropriate concentration and for an appropriate length of time which can easily be determined by a skilled person using routine techniques, and will depend on such factors as the particular photosensitizing agent used, the mode of administration, the course of treatment, the age and weight of the patient/subject, the medical indication, the body or body area to be treated and may be varied or adjusted according to choice.
  • concentration of the photosensitizing agent is conveniently such that once taken up into the cell, e.g. into, or associated with, one or more of its intracellular compartments and activated by irradiation, one or more cell structures are disrupted e.g. one or more intracellular compartments are lysed or disrupted.
  • photosensitizing agents as described herein may be used at a concentration of for example 0.1 to 50 ⁇ g/ml.
  • the range can be much broader, e.g. 0.0005-500 ⁇ g ml.
  • the photosensitizing agent may be used in the range 0.05-20 mg/kg body weight when administered systemically.
  • a range of 0.005-20mg/kg body weight may be used for systemic administration.
  • the total dose provided may be in the order of 1 -5000 ⁇ 9, for example 10-2500, 25-1000, 50-500, 10-300, 25-200, or 100-300 ⁇ 9.
  • the dose is selected from 100 ⁇ g, 150 ⁇ g, 200 ⁇ g and 250 ⁇ g.
  • the dose is 75-125 ⁇ g, e.g. 100 ⁇ g.
  • the dose may be in the region of 0.001 - 500 ⁇ g or 0.1 -500 ⁇ g, for example 0.001 -0.1 , 0.0025-1 , 0.01 -50, 0.0025-250, 1 -250, 2.5-100, 2.5-40, 5-50, 1 -30 or 10-3( ⁇ g.
  • the dose is selected from ⁇ g, ⁇ g, 20 ⁇ g and 25 ⁇ g.
  • the dose is 7.5-12.5 ⁇ g, e.g.
  • the doses provided are for a human of average weight (i.e. 70kg).
  • the photosensitizer dose may be dissolved in 100 ⁇ -1 ml, i.e. the concentration may be in the range of 0.01 - 50000 ⁇ g/ml or 1 -50000 ⁇ g/ml. In smaller animals the concentration range may be different and can be adjusted accordingly though when administered locally, little variation in dosing is necessary for different animals.
  • concentration of the antibacterial agent as defined herein will also depend on the particular molecule which is to be used, the mode of administration, the course of treatment, the age and weight of the patient/subject, the medical indication, the body or body area to be treated and may be varied or adjusted according to choice.
  • antibacterial agents as described herein may be used at a concentration of for example 0.01 to 50 ⁇ g/ml.
  • the range can be much broader, e.g. 0.0005-500 ⁇ g/ml.
  • the antibacterial agent may be used in the range 0.05-100 mg/kg body weight when administered systemically.
  • a range of 0.005-1 OOmg/kg body weight, preferably 0.1 to 50mg/kg, may be used for systemic administration.
  • the dose may be in the region of 1 -50000 ⁇ g, for example 10-25000, 25-10000, 50-5000, 10-3000 or 100-3000 ⁇ g.
  • the dose is selected from 1 mg, 1 .5mg, 2mg and 2.5mg.
  • the dose is 0.75-1 .25 mg, e.g. 1 mg.
  • the doses provided are for a human of average weight (i.e. 70kg).
  • the antibacterial agent dose may be dissolved in 100 ⁇ -1 ml, i.e. the concentration may be in the range of 1 -50000 ⁇ g/ml. In smaller animals the concentration range may be different and can be adjusted accordingly though when administered locally, little variation in dosing is necessary for different animals.
  • the photosensitizing agent and the antibacterial agent are administered together, but this may be varied. Thus different times or modes or sites of administration (or contact with the cell) are contemplated for each of the different components and such methods are encompassed within the scope of the invention.
  • the antibacterial agent as defined herein is administered separately from the photosensitizing agent, for example in a separate formulation, or systemically, e.g. via oral administration.
  • the antibacterial agent or the photosensitizing agent may be administered prior to administration of the photosensitizer or antibacterial agent, respectively, for example up to 24 or 48 hours before.
  • the separate administrations are separated by less than 48, 24, 12, 8, 4 or 2 hours.
  • the photosensitizer may be administered first (e.g. locally to avoid skin photosensitivity) and the antibacterial agent may then be administered systemically.
  • the contact between the cell(s) and the photosensitizing agent and/or antibacterial agent as defined herein is conveniently from 15 minutes to 24 (or 48) hours (e.g. 15 or 30 minutes to 4 hours, e.g. 1 -2 hours).
  • the contacting may be simultaneous or sequential or the timing of contacting with the separate components may overlap.
  • the contacting step refers to the total contact time of the cell(s) with the agent in question and that contacting time may be made up of a number of discrete separate contacting steps.
  • the agent may be removed from contact with the cell(s) for a period of time before the irradiation step.
  • the contacting step for each agent may be 15 (or 30) to 120 minutes.
  • the cells may not be contacted with the agents immediately after administration (e.g. where systemic administration is used) and in such case the agents will need to be administered sufficiently before illumination so that the agents reach the target cells and have contact with those cells for the required contact time, as discussed hereinbelow in more detail.
  • the initial incubation of the cell is with the photosensitizing agent.
  • the time between the administration of the photosensitizing agent and the antibacterial agent is a matter of hours.
  • the photosensitizing agent may be applied 16 to 20 (or 40 to 44) hours, e.g. 18 hours, before illumination
  • the antibacterial agent may be applied 1 to 3 hours, e.g. 2 hours before illumination.
  • the time between the administration of the photosensitizing agent and the antibacterial agent may be in the range of 15 to 23 (or 47) hours. This timing applies regardless of which agent is administered first.
  • the cell(s) may be placed into photosensitizer/antibacterial agent-free medium after the contact with the photosensitizer/antibacterial agent and before irradiation, e.g. for 30 minutes to 4 hours, e.g. from 1 .5 to 2.5 hours, depending on the timing of the incubation with the photosensitizer and antibacterial agent.
  • an appropriate method and time of incubation by which the various agents are brought into contact with the target cells will be dependent on factors such as the mode of administration and the type of agents which are used. For example, if the agents are injected into a tissue or organ which is to be
  • the cells near the injection or application point will come into contact with and hence tend to take up the agents more rapidly than the cells located at a greater distance from the injection or application point, which are likely to come into contact with the agents at a later time point and lower concentration.
  • a time of 6-24 (or 6-48) hours may be used.
  • agents administered by intravenous injection or orally may take some time to arrive at the target cells and it may thus take longer post- administration e.g. several days, in order for a sufficient or optimal amount of the agents to accumulate in a target cell or tissue.
  • the time of administration required for individual cells in vivo is thus likely to vary depending on these and other parameters.
  • the time at which the molecules come into contact with the target (i.e. infected) cells must be such that before irradiation occurs an appropriate amount of the photosensitizing agent has been taken up by the target cells and either: (i) before or during irradiation the antibacterial agent has either been taken up, or will be taken up after sufficient contact with the target cells, into the cell, for example into the same or different intracellular compartments relative to the photosensitizing agent or (ii) after irradiation the antibacterial agent is in contact with the cells for a period of time sufficient to allow its uptake into the cells.
  • any mode of administration common or standard in the art may be used, e.g. oral, parenteral (e.g. intramuscular, transdermal, subcutaneous, percutaneous, intraperitoneal, intrathecal or intravenous), intestinal, buccal, rectal or topical, both to internal and external body surfaces etc.
  • parenteral e.g. intramuscular, transdermal, subcutaneous, percutaneous, intraperitoneal, intrathecal or intravenous
  • intestinal, buccal, rectal or topical both to internal and external body surfaces etc.
  • the invention can be used in relation to any tissue which contains cells to which the photosensitizing agent containing compound or the antibacterial agent is localized, including body fluid locations, as well as solid tissues. All tissues can be treated as long as the photosensitizer is taken up by the target cells, and the light can be properly delivered.
  • Preferred modes of administration are intradermal, subcutaneous or topical administration or injection.
  • administration is topical (also referred to herein as local administration).
  • Administration may be by application of the required agents to the cell(s) at the time of the treatment/prevention method (e.g. administration to the
  • the antibacterial agent and/or the photosensitizing agent may be provided on or within (e.g.
  • Such agents may be released slowly with time or released on demand and the treatment /prevention step initiated by irradiation (optionally with administration of one of the required agents if not present in/on the biomaterial). Conveniently in assessing timings and doses such administration is considered local administration.
  • the methods or parts thereof may be repeated.
  • the method in its entirety may be performed multiple times (e.g. 2, 3 or more times) after an appropriate interval or parts of the method may be repeated, e.g. further administration of the antibacterial agent and/or photosensitizing agent as defined herein or additional irradiation steps.
  • the method or part of the method may be performed again a matter of days, e.g. between 5 and 60 days (for example 7, 14, 15, 21 , 22, 42 or 51 days), e.g. 7 to 20 days, preferably 14 days, or weeks, e.g. between 1 and 5 weeks (for example, 1 , 2, 3 or 4 weeks) after it was first performed. All or part of the method may be repeated multiple times at appropriate intervals of time, e.g. every two weeks or 14 days. In a preferred embodiment the method is repeated at least once. In another embodiment the method is repeated twice.
  • “Irradiation” to activate the photosensitizing agent refers to the administration of light directly or indirectly as described hereinafter.
  • the cell(s) which may be present in a subject
  • Illumination of the cell or subject may occur approximately 15 minutes to 24 (or 48) hours after administration of the various components for use in the method as defined herein.
  • illumination may occur from, for example 15 to 120 minutes after administration, whereas in in vivo methods a longer time after administration may be required if contact time with the target cells within the subject is not immediate (depending on the route of administration), e.g. from 15 minutes to 24 (or 48) hours after administration.
  • the light irradiation step to activate the photosensitizing agent may take place according to techniques and procedures well known in the art.
  • the dose, wavelength and duration of the illumination must be sufficient to activate the photosensitizing agent, i.e. to generate reactive species.
  • the wavelength of light to be used is selected according to the
  • Suitable artificial light sources are well known in the art, e.g. using blue (400-475nm) or red (620-750nm) wavelength light.
  • TPCS 2a for example, a wavelength of between 400 and 500nm, more preferably between 400 and 450nm, e.g. from 430-440nm, and even more preferably approximately 435nm, or 435nm may be used.
  • photosensitizer e.g. a porphyrin or chlorin
  • may be activated by green light for example the KillerRed (Evrogen, Moscow, Russia) photosensitizer may be activated by green light.
  • Suitable light sources are well known in the art, for example the LumiSource® lamp of PCI Biotech AS.
  • an LED-based illumination device which has an adjustable output power of up to 60mW and an emission spectra of 430-435nm may be used.
  • a suitable source of illumination is the PCI Biotech AS 652nm laser system SN576003 diode laser, although any suitable red light source may be used.
  • the time for which the cells are exposed to light in the methods of the present invention may vary. The efficiency of the internalisation of a molecule into the cytosol increases with increased exposure to light to a maximum beyond which cell damage and hence cell death increases.
  • a preferred length of time for the irradiation step depends on factors such as the target, the photosensitizer, the amount of the photosensitizer accumulated in the target cells or tissue and the overlap between the absorption spectrum of the photosensitizer and the emission spectrum of the light source.
  • the length of time for the irradiation step is in the order of seconds to minutes or up to several hours (even up to 12 hours), e.g. preferably up to 60 minutes e.g. from 0.25 or 1 to 60 minutes, e.g. from 5 to 60 minutes, preferably for 10 to 20 minutes, preferably for 15 minutes.
  • Shorter irradiation times may also be used, for example 1 to 60 seconds, e.g. 10-50, 20-40 or 25-35 seconds, e.g. when higher doses of photosensitizing agent are used.
  • Appropriate light doses can be selected by a person skilled in the art and again will depend on the photosensitizer used and the amount of photosensitizer accumulated in the target cell(s) or tissues.
  • the light doses are usually lower when photosensitizers with higher extinction coefficients (e.g. in the red area, or blue area if blue light is used, depending on the photosensitizer used) of the visible spectrum are used.
  • a light dose in the range of 0.24 - 7.2J/cm 2 at a fluence range of 0.05-20 mW/cm 2 e.g.
  • 2.0 mW/cm 2 may be used when an LED-based illumination device which has an adjustable output power of up to 60mW and an emission spectra of 430-435nm is employed.
  • the LumiSource® lamp a light dose in the range of 0.1 -6J/cm 2 at a fluence range of 0.1 - 20 (e.g. 13 as provided by Lumisource®) mW/cm 2 is appropriate.
  • a light dose of 0.03-1 J/cm 2 e.g. 0.3J/cm 2
  • at a fluence range of 0.1-5 mW/cm 2 e.g. 0.81 mW/cm 2
  • 0.03-1 J/cm 2 e.g. 0.3J/cm 2
  • a fluence range of 0.1-5 mW/cm 2 e.g. 0.81 mW/cm 2
  • the methods of the invention may inevitably give rise to some cell damage by virtue of the photochemical treatment i.e. by photodynamic therapy effects through the generation of toxic species on activation of the photosensitizing agent.
  • this cell death may not be of consequence and may indeed be advantageous to remove some bacteria-infected cells. In most embodiments, however, cell death is avoided, e.g. to allow the antibacterial effects to occur.
  • the methods of the invention may be modified such that the fraction or proportion of the surviving cells is regulated by selecting the light dose in relation to the concentration or dose of the photosensitizing agent. Again, such techniques are known in the art.
  • substantially all of the cells, or a significant majority are not killed (of those subject to the treatment).
  • In vitro cell viability following PCI treatment can be measured by standard techniques known in the art such as the MTS test.
  • In vivo cell death of one or more cell types may be assessed within a 1 cm radius of the point of administration (or at a certain depth of tissue), e.g. by microscopy. As cell death may not occur instantly, the % cell death refers to the percent of cells which remain viable within a few hours of irradiation (e.g. up to 4 hours after irradiation) but preferably refers to the % viable cells 4 or more hours after irradiation.
  • PCI allows the antibacterial agent to be released into the cytosol of the cell(s). The agent may then interact with the bacteria to kill or damage the bacteria or to prevent its replication.
  • “Kill” refers to destruction of a bacteria to the extent that no further replication can take place.
  • “Damage” refers to affecting the bacteria's ability to function normally, such that it may die or be unable to replicate.
  • Preventing replication refers to prevention of the replication of the bacteria partially or completely, e.g. according to the percentages described hereinafter.
  • a method, treatment or use described herein results in the death or damage of at least 25, 50, 75 or 90% of the bacteria to which the treatment is applied or prevents replication such that a bacterial infection is prevented or reduced, e.g. by at least 30, 40, 50, 60, 70, 80 or 90% relative to a control to which the treatment is not applied.
  • the method may be performed in vivo, in vitro or ex vivo. Preferably the method is used in vivo.
  • the method of the invention may alternatively be described as an in vitro method of killing, damaging or preventing the replication of a bacteria in a cell(s), comprising contacting the cell(s) with an antibacterial agent and a photosensitizing agent and irradiating the cell(s) with light of a wavelength effective to activate the photosensitizing agent, wherein the antibacterial agent is released into the cytosol of the cell(s) and kills, damages or prevents the replication of bacteria in said cell(s).
  • Preferred aspects described above also apply to this method.
  • the subject refers to a mammal, reptile, bird, insect or fish.
  • the subject is a mammal, particularly a primate (preferably a human), domestic or companion animal, livestock or laboratory animal.
  • preferred animals include mice, rats, rabbits, guinea pigs, cats, dogs, monkeys, pigs, cows, goats, sheep and horses.
  • the photosensitizing agent and the antibacterial agent may be provided in a composition.
  • they may be in separate solutions or compositions allowing different mechanisms or timings for administration or application.
  • co-administration and co-application refers to use of both components in the same method rather than simultaneous use (either in terms of timing or in the same composition).
  • the invention also provides an antibacterial agent as defined hereinbefore, and a photosensitizing agent as defined hereinbefore or a composition comprising an antibacterial agent and a photosensitizing agent as defined hereinafter for use in treating an intracellular bacterial infection in a subject, wherein preferably said use comprises a method as defined hereinbefore.
  • the invention also provides use of an antibacterial agent as defined hereinbefore and/or a photosensitizing agent as defined hereinbefore in the manufacture of a medicament for treating an intracellular bacterial infection in a subject, preferably by a method as defined hereinbefore.
  • an antibacterial agent and a photosensitizing agent as an antibacterial, wherein preferably the agents are as described hereinbefore and used in accordance with the method described herein.
  • the invention further provides a composition comprising an antibacterial agent as defined hereinbefore and a photosensitizing agent as defined
  • composition may be in the form of a pharmaceutical composition comprising in addition one or more pharmaceutically acceptable diluents, carriers or excipients.
  • compositions and products of the invention, described hereinafter may be formulated in any convenient manner according to techniques and procedures known in the pharmaceutical art, e.g. using one or more
  • compositions may be formulated as slow or delayed release compositions.
  • “Pharmaceutically acceptable” as referred to herein refers to ingredients that are compatible with other ingredients of the compositions (or products) as well as physiologically acceptable to the recipient.
  • the nature of the composition and carriers or excipient materials, dosages etc. may be selected in routine manner according to choice and the desired route of administration, purpose of treatment etc. Dosages may likewise be determined in routine manner and may depend upon the nature of the molecule (or components of the composition or product), purpose of treatment, age of patient/subject, mode of administration etc. In connection with the photosensitizing agent, the potency/ability to disrupt membranes on irradiation, should also be taken into account.
  • the invention also provides a product comprising an antibacterial agent as defined hereinbefore, and a photosensitizing agent as defined hereinbefore as a combined preparation for simultaneous, separate or sequential use in treating an intracellular bacterial infection in a subject.
  • kits for use in treating an intracellular bacterial infection in a subject comprising
  • the products and kits of the invention may be used to treat or prevent intracellular bacterial infection as defined hereinbefore.
  • Figure 1 shows the effect of PCI on efficacy of gentamicin treatment of intracellular S. epidermidis in Raw 264.7 cells. The initial numbers of intracellular S.
  • epidermidis were determined to be 10 6 CFU/well immediately after phagocytosis (Uptake). After phagocytosis, cells were treated with 0.25 ⁇ g/ml TPPS 2a only, with 1 , 10 and 30 ⁇ g/ml gentamicin (GEN) alone (-), or with the respective gentamicin- TPPS 2a combinations (+) for 2 hours. Subsequently the cells were illuminated for 10 or 15 minutes. Non-illuminated cells (no illumination) and non-treated, illuminated cells (No GEN, TPPS 2 a "-”) were used as controls.
  • GEN gentamicin
  • Figure 2 shows the intracellular distribution of gentamicin and TPCS 2 a in Raw 264.7 cells with and without illumination of 2 minutes to activate the photosensitizer.
  • Cells were incubated overnight in culture medium containing 10 ⁇ g/ml gentamicin (GEN, blue fluorescent) and 1 ⁇ g/ml TPCS 2 a (red fluorescent) before illumination.
  • Figure 3 shows determination of the doses of S. aureus for zebrafish embryo infection and visualization of co-localization of S. aureus and zebrafish phagocytic cells.
  • Figure 4 shows percent survival of S. aureus-infected embryos treated with 0.4 ng gentamicin (GEN), 0.1 and 0.05 ng GEN alone or combined with 2.5 * 10 3 ng TPCS 2a (T).
  • PBS mock treatment was used as control.
  • Initial group size ranged from 31 to 33 embryos.
  • Differences between survival of gentamicin alone or gentamicin-TPCS2a treatment groups and survival of the PBS mock treatment group, as well as between gentamicin-TPCS 2a groups and the respective gentamicin only groups were analyzed using Log-rank test. ** p ⁇ 0.01. *** p ⁇ 0.001.
  • Figure 5 shows PCI re-localises vancomycin from endocytic vesicles to the cytosol in a macrophage cell line.
  • the intracellular localization of TPCS 2a and BODIPY® FL-Vancomycin was analysed by fluorescence microscopy before (A) and after (B) illumination.
  • FIG. 6 shows PCI induces disaggregation of vancomycin inside macrophages.
  • RAW 264.7 cells were treated in the same was as cells for Figure 5.
  • the microscopy light excitation time was the same for the samples both before and after PCI, so that the fluorescence intensity can be quantitatively compared.
  • Figure 7 shows percent survival of S. aureus-infected embryos treated with 0.4 ng vancomycin (Vanco) alone or combined with 2.5 * 10 3 ng TPCS 2a and illumination (T). PBS mock treatment was used as control. The difference between survival of vancomycin alone group and vancomycin-TPCS 2a /illumination group was analyzed using Log-rank test, * p ⁇ 0.05.
  • EXAMPLE 1 ENHANCED ANTIBACTERIAL EFFICACY OF GENTAMICIN AGAINST INTRACELLULAR STAPHYLOCOCCAL INFECTION BY PCI
  • TPPS 2a tetraphenyl porphyrin disulphonate
  • TPCS 2a tetraphenyl chlorin disulphonate
  • TPPS 2a and TPCS 2a possess very similar physico- chemical properties, but only TPCS 2a can be activated by red light. Red light has very good tissue penetration and hence TPCS 2a is suitable for broad clinical applications.
  • the aim was to assess whether PCI combined with antibiotics (antibacterial agents) could combat intracellular bacterial infection by enhancing cytosolic delivery of antibiotics upon illumination. Gentamicin was selected to study the potential effect of PCI since it has low intracellular antimicrobial efficacy.
  • S. epidermidis strain 0-47 ( Riool et al., 2014, supra) was used for in vitro studies with Raw 264.7 mouse macrophages (Raw 264.7 cells) (Xia et al., 2008, Acs Nano, 2(10), p2121 -2134).
  • FCS fetal calf serum
  • aureus strains ATCC49230 was used for zebrafish embryo infection.
  • S. aureus strain RN4220 containing GFP expression plasmids WVW189 (S. aureus RN4220- GFP) was constructed, following the protocol described earlier (Riool et al., 2014, supra; and Riool et al., 2017, European Cells & Materials, 33, p143-157), and used for / ' n vivo visualization of cell-bacteria interaction in zebrafish embryos.
  • the bacterial suspensions (in PBS or RPMI) with desired concentrations for different experiments were prepared, following the protocol described earlier (Riool et al., 2014, supra; and Riool et al., 2017, supra).
  • Raw 264.7 cells were seeded in a 96-well plate (polystyrene NunclonTM clear TC plate, flat bottom, Greiner, The Netherlands) at a concentration of 1x10 5 cells/well and incubated overnight in RPMI at 37 °C in a humidified atmosphere containing 5 % C0 2 (unless specified otherwise).
  • the cells were subsequently incubated overnight in 200 ⁇ of RPMI containing gentamicin (15.6 to 1000 ⁇ g/ml), or incubated for 2 hours in RPMI containing photosensitizer TPPS2a (0.1 to 0.4 ⁇ g/ml) (PCI Biotech AS, Norway) which was replaced with refresh RPMI in order to remove unbound TPPS 2a before incubation of another 2 hours.
  • Raw 264.7 cells incubated in RPMI all the time were used as controls. The cells were protected from light using aluminium foil apart from illumination of 15 minutes using
  • Raw 264.7 cells were incubated in 200 ⁇ of RPMI either containing gentamicin alone (1 , 10 or 30 ⁇ g/ml) or combined with TPPS 2a (0.25 ⁇ 9 ⁇ ) for 2 hours, with non-treated cells incubated in PMI or RPMI only containing TPPS 2 a as controls.
  • TPPS 2 a medium was replaced with refresh RPMI containing gentamicin with the identical concentrations before incubation of another 2 hours in order to remove unbound TPPS 2a - Cells were then incubated in refresh RPMI containing 1 ⁇ g/ml gentamicin, illuminated for 10 or 15 minutes with non-illuminated cells as controls. After illumination, cells were incubated overnight, lysed before quantitative culture of surviving bacteria.
  • gentamicin in K 2 CO 3 , pH 9 (Sigma-Aldrich) was mixed with Alexa Fluor 405 succinimidyl ester (Life Technologies) to minimize the possibility of labelling individual gentamicin molecules with more than one Alexa Fluor 405 molecule.
  • the reaction mixture was separated by reversed phase chromatography using a C-18 column to purify the conjugate from unconjugated gentamicin and Alexa Fluor 405 molecules.
  • the isolated Alexa Fluor 405 labelled gentamicin was aliquoted, lyophilized, and stored in the dark at -20°C until used.
  • Toxicity test of gentamicin alone or combined with TPCS 2a in zebrafish embryos Gentamicin or TPCS 2 a solution (both in PBS) or mixtures were injected into WT zebrafish embryos at 32 hours post fertilization (hpf). PBS was injected into control embryos. The embryos were protected from light using aluminium foil apart from illumination for 10 minutes using LumiSource, at 34 hpf. Survival of embryos (heartbeat, movement) was monitored daily until 6 dpi.
  • Wild type zebrafish embryos were injected with graded inocula of S. aureus ATCC 49230 at 30 hpf, and individually maintained in 200 ⁇ of E3 medium. Medium was refreshed daily. The injected doses were checked by quantitative culture of 5-6 embryos per group, crushed using a MagNA lyser (Roche, The Netherlands). Survival was monitored daily until 4 dpi.
  • Wild type zebrafish embryos were intravenously injected with a suitable dose of S aureus ATCC 49230 via the blood island at 30 hpf, and randomly divided into groups for different treatments.
  • a suitable dose of S aureus ATCC 49230 via the blood island at 30 hpf, and randomly divided into groups for different treatments.
  • 32 hpf 1 nl of PBS solution containing gentamicin alone (0.05, 0.1 or 0.4 ⁇ g/ml) or combined with 0.25 ⁇ g/ml TPCS 2 a (to provide 2.5x10 3 ng) was intravenously injected.
  • Control embryos received PBS injections. The embryos were protected from light using aluminium foil apart from illumination of 10 minutes, at 34 hpf, and separately maintained in E3 medium, which was refreshed daily. Survival was monitored until 6 dpi.
  • Raw 264.7 cells were treated with TPPS 2 a alone (0.25 ⁇ g/ml) or with TPPS 2a in presence of S. epidermidis (TPPS 2a + S.
  • TPPS 2a -PCI significantly enhanced the killing by 30 ⁇ g/ml gentamicin (1 log reduction of the numbers of intracellular bacteria), but not by 10 ⁇ g/ml gentamicin.
  • killing of intracellular S. epidermidis in Raw 264.7 cells treated with 10 and 30 ⁇ g/ml gentamicin combined with TPPS 2a was significantly increased to 1 and 2.5 log reduction, respectively.
  • TPCS 2 a absorbs red light and therefore is suitable for applications in vivo.
  • TPCS 2a was selected for this cell study and in vivo studies with zebrafish embryos. Without illumination, both gentamicin and TPCS 2 a localized within cellular compartments in the periphery of the cells, likely endocytic vesicles. After illumination both agents were released into the cytosol. Gentamicin seemed to accumulate at the nuclei of the Raw 264.7 cells.
  • Toxicity of gentamicin and TPCS 2a alone or in combination to zebrafish embryos To test their toxicity to zebrafish embryos, the effect of injection of graded doses of gentamicin (Doses ranging from 0.16 to 16 ng per 1 nl of PBS were injected per embryo, using 1 nl of PBS as control), TPCS 2a (Doses of 2.5 * 10 "2 , 2.5 * 10 "3 and 2.5 * 10 "4 ng per 1 nl of PBS were injected per embryo, using 1 nl PBS as control) and gentamicin-TPCS 2a combinations (Doses of 1.6 and 0.8 ng GEN alone or combined with 2.5 * 10 "3 ng TPCS 2a (in 1 nl of PBS) were injected, using 1 nl of PBS injection as control) on survival was assessed.
  • graded doses of gentamicin Doses ranging from 0.16 to 16 ng per 1 nl of PBS were injected per embryo, using
  • PCI has been shown to enhance intracellular activity of gentamicin, an antibiotic with limited antimicrobial efficacy inside cells, against Staphylococci both in vitro and in vivo.
  • PCI induced cytosolic release of gentamicin after illumination and increased eradication of phagocytosed S. epidermidis.
  • PCI enhanced the efficacy of gentamicin treatment against (intracellular) S. aureus infection and lowered the required dose.
  • the quantity of antibiotics accumulating inside cells is essential for the efficiency of killing of intracellular bacteria (Serai et ai, 2003, supra; and Barcia-Macay et ai, 2006, Antimicrob. Agents Ch., 50(3), p841 -851 ).
  • the cells were exposed to relatively high concentrations of gentamicin (10 and 30 ⁇ g/ml) for 2 hours. Even with such high extracellular concentrations, gentamicin did not kill the intracellular S. epidermidis bacteria. Strikingly, combining the treatment with the use of PCI significantly improved the antimicrobial efficacy of gentamicin ( Figure 1 ).
  • PCI can be expected also to improve the intracellular activity of other antibiotics such as vancomycin, oritavancin and various macrolides. This consequently may prevent resistance development due to low, permissive concentrations of such antibiotics inside cells. PCI therefore can increase the number of antibiotics which can successfully treat intracellular infection. In addition, the required doses of antibiotics may be reduced using PCI.
  • PCI may be used to improve the antibiotic treatment of intracellular infection and help prevent resistance development.
  • EXAMPLE 2 ENHANCED ANTIBACTERIAL EFFICACY OF VANCOMYCIN AGAINST INTRACELLULAR STAPHYLOCOCCAL INFECTION BY PCI
  • PCI re-localises vancomycin from endocytic vesicles to the cytosol in macrophages.
  • the macrophage cell line RAW 264.7 was incubated with 1 ⁇ g/ml TPCS 2 a and 50 ⁇ g/mL BODIPY® FL-Vancomycin (Life Technologies) (Vanco-FL) for 18 h. The cells were washed 2 times in drug-free medium and incubated for 4 h in drug-free medium before illumination with LumiSource for 120 s. The intracellular localization of TPCS 2 a and BODIPY® FL-Vancomycin was analysed by fluorescence microscopy before (A) and after (B) illumination using the following filter settings: TPCS 2a : Excitation: Band-pass: 395-440 nm, dichroic beam splitter 460 nm. Emission: Long-pass 620 nm. BODIPY: Excitation: Band-pass: 450-490 nm.
  • vancomycin from endocytic vesicles to the cell cytosol also seems to induce a strong increase in the fluorescence signal from the vancomycin-BODIPY.
  • the probable reason for this is that the vancomycin molecules are aggregated inside the endosomes and they disaggregate upon release into the much larger distribution volume in the cytosol. It is well known that aggregation of fluorescent molecules leads to quenching of the fluorescence, and that the fluorescence from fluorophores in aggregates will increase substantially upon disaggregation. Since aggregated molecules in general will be unable to interact with therapeutic targets the disaggregation seen after PCI will add further to the enhancement of antimicrobial activity that can be achieved with PCI.
  • PCI enhances the anti-microbial effect of vancomycin in zebrafish embryos.
  • the median CFU number of injected S. aureus was determined to be 4300

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